The objective of this project is to develop an innovative molecular genetic approach to analyze the allelic status in plasma for cancer detection. Allelic imbalance (AI), defined as losses or gains of particular chromosomal regions, is a unique genetic abnormality in almost all human cancers. Since tumors release substantial amounts of tumor DNA into the systemic circulation, AI in plasma DNA may represent a universal marker in cancer patients. Traditional methods to determine AI in plasma or serum using micro satellite markers are fraught with a variety of technical difficulties such as contamination of normal DNA, variable degradation of plasma DNA and difficulty for quantitation and interpretation. To overcome these obstacles, we propose to employ our recently developed PCR-based approach called Digital Single Nucleotide Polymorphism (SNP) analysis in which the paternal or maternal alleles within a plasma DNA sample are individually counted thus allowing a quantitative measure of such imbalance in the presence of normal DNA. With this technique, the alleles present in plasma can be directly counted to determine whether there is an imbalance. This R21/R33 combined proposal consists of two phases. The R2t phase is to develop Digital SNP analysis suitable for analysis using plasma DNA. The specific aims are: 1) select SNP markers for Digital SNP analysis in plasma; 2) optimize the conditions for Digital SNP analysis; and 3) improve the automation and throughput. The R33 phase is to test the feasibility of our innovated technology for practical application. The specific aims are: 1) demonstrate allelic imbalance in plasma DNA of cancer patients using Digital SNP analysis; and 2) assess the clinical potential of Digital SNP analysis for detection of asymptomatic cancer. In conclusion, the results generated from this proposal will provide a clear measurement of tumor-released DNA into circulation and whether tumor-released DNA can be detected by an efficient approach for a potential cancer screening. Thus, the encouraging results from this study will ensure the detection of AI in plasma a promising cancer research direction for further exploration.